The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is expressed in many epithelia and in the heart. Phosphorylation of CFTR by protein kinases is thought to be an absolute prerequisite for the opening of CFTR channels. In addition, nucleoside triphosphates were shown to regulate the opening of phosphorylated CFTR. Here, we report that phosphatidylinositol 4,5-bisphosphate (PIP(2)) activates human CFTR, resulting in ATP responsiveness of PIP(2)-treated CFTR. PIP(2) alone is not sufficient to open CFTR, but ATP opens nonphosphorylated CFTR after application of PIP(2). The effect of PIP(2) is independent of protein kinases, as PIP(2) activates CFTR in the complete absence of Mg. Phosphatidylinositol and phosphatidylinositol monophosphate activate CFTR less efficiently than PIP(2). PIP(2) application to phosphorylated CFTR may inhibit the CFTR chloride current. We suggest that regulation of CFTR by PIP(2) is a previously unrecognized, alternative mechanism to control chloride conductance.